Quantitative Cu-Homeostasis in Live Mammalian Cells at the Single-Molecule Level

项目摘要/摘要 了解金属蛋白的响应机制是阐明铜的生物功能的关键 并找出金属稳态异常引起的疾病的原因。细胞对铜的摄取和 分泌需要相关金属蛋白以时空协调的方式进行调节，以确保适当的 细胞内铜水平。然而，在铜领域，人们对金属蛋白是如何单独调节的知之甚少 也不是在它们的自然环境中，即在细胞中，系统地相互合作。我们的研究目标是 了解哺乳动物活细胞对铜摄取和分泌金属蛋白的反应机制， 特别关注金属蛋白如何调节它们的行为，如空间分布、低聚状态、 蛋白质之间和结构域之间的相互作用，以时空定义的方式实现适当的铜平衡。上一首 PI的成就包括发现MERR家族金属调节剂调节的新机制 通过检测蛋白质-DNA相互作用实现外排泵的转录和铜响应的动态组装 和蛋白质在活细菌中的扩散行为的单分子超分辨率显微镜。利用 这些技术的力量，结合最近开发的活细胞单分子荧光- 通过共振能量转移实验，我们将阐明金属蛋白在摄取过程中的响应机制 以及活的哺乳动物细胞中的分泌途径。使用CTR1和ATOX1-ATP7A/B作为初始示例 摄取和分泌金属蛋白，拟议的实验将(1)量化铜依赖的寡聚体状态 (2)确定ATOX1与铜的优先相互作用 ATP7A和ATP7B以及ATP7B突变如何影响细胞铜防御中的铜稳态 多能干细胞来源于肝细胞。除了单分子超分辨的主要方法 荧光成像技术、互补的整体光谱和生化测量将是 比较一下。通过与铜稳态和铜平衡专家的合作，该研究计划得到了进一步的加强 干细胞领域。这项研究意义重大，因为它将提供对金属蛋白的机械洞察- 同步辐射X射线介导的铜摄取和分泌过程及其互补信息 细胞内铜再分布的荧光研究。人诱导多能干细胞的比较研究 来自细胞(HiPSC)的健康和患病肝细胞将了解疾病突变如何扰乱细胞铜 平衡，为制定威尔逊病的治疗策略提供知识基础。这项研究是 创新，因为它通过将重点转移到定义响应来代表对现状的实质性改变 利用包括单分子超分辨在内的先进方法研究金属蛋白的机制 显微镜和HiPSC来源的肝细胞。活细胞成像方法也绕过了一般的 研究膜复合体的挑战，其体外重建技术要求很高。HiPSC- 来源的病变肝细胞为研究肝豆状核变性的发病机制提供了理想的平台。

PROJECT SUMMARY/ABSTRACT Understanding responsive mechanisms of metalloproteins is key to elucidate biological functions of copper (Cu) and to identify the causes of diseases resulting from abnormal metal homeostasis. The cellular Cu uptake and secretion require relevant metalloproteins to adjust in a spatiotemporally coordinated manner to assure proper cellular Cu level. However, in the Cu field, little is known about how metalloproteins are individually regulated nor systematically cooperate with each other in their native environment, i.e., in cells. Our research goal is to understand the responsive mechanisms of Cu-uptake and secretory metalloproteins in live mammalian cells, with specific focuses on how metalloproteins adjust their behaviors such as spatial distributions, oligomeric states, inter-protein and inter-domain interactions for proper Cu balance in a spatiotemporally defined manner. Previous achievements of the PI include discoveries of novel mechanisms of MerR-family metalloregulators in regulating transcription and Cu-responsive dynamic assembly of efflux pumps by examining the protein-DNA interaction and protein diffusive behaviors in live bacteria using single-molecule super-resolution microscopy. Leveraging the power of these technologies, in combined with the recently developed live-cell single-molecule fluorescence- resonance-energy-transfer assay, we will elucidate the responsive mechanisms of metalloproteins in the uptake and secretory pathways in live mammalian cells. Using CTR1 and ATOX1-ATP7A/B as the initial examples of uptake and secretory metalloproteins, the proposed experiments will (1) quantify Cu-dependent oligomeric state distribution and identify the Cu-responsive moiety of CTR1; (2) define the preferential interaction of ATOX1 to ATP7A and ATP7B and how mutations in ATP7B affect Cu homeostasis in cellular Cu defending using induced pluripotent stem cells derived hepatocytes. In addition to primary approaches of single-molecule super-resolution fluorescence imaging techniques, complementary bulk spectroscopic and biochemical measurements will be compared. The research program is further enhanced by collaborations with the experts in Cu homeostasis and stem cell fields. The research is significant because it will provide mechanistic insights into metalloprotein- mediated Cu-uptake and secretion processes as well as complementary information for synchrotron X-ray fluorescence studies on intracellular Cu-redistribution. The comparison between human induced pluripotent stem cell (hiPSC)-derived healthy and diseased hepatocytes will inform how disease mutations disrupt cellular Cu balance, providing the knowledge base to devise therapeutic strategies for Wilson's diseases. The research is innovative because it represents a substantive departure from the status quo by shifting focus to define response mechanisms of metalloproteins using advanced approaches including single-molecule super-resolution microscopy and hiPSC-derived hepatocytes. The live-cell imaging approach also circumvents the general challenge in studying membrane complexes, whose in vitro reconstitution is technically demanding. The hiPSC- derived diseased hepatocytes provide an ideal platform to study the pathogenesis of Wilson's disease.